The present invention relates to an immobilized microbial consortium and a process for the preparation of the said microbial consortium useful for the treatment of phenolic waste-water.
Phenols are hydroxy derivatives of benzene which originate both from anthropogenic sources such as forest fire, decay of lignocellulosic materials as well as from various human activities which originate from pharmaceutical industries, pesticide industries, petroleum refineries and wood processing industries.
In petroleum refineries, crude petroleum passes through various stages of refining before it is finally used as a fuel. The effluent of petroleum refineries contains a large number of compounds, most of which are hydrocarbons. In addition to the hydrocarbons, the effluent also contains grease (0.01 to 2%), sulphur (0-6%), cyanide (0.1 to 0.2%), suspended solids etc. (Khan 1995, Abdulbasher, 1985).
Phenols are by-products obtained during the refining process. The concentration of phenols in the waste-waters depends largely on the nature of the crude oil processed. Phenol when present in waste-waters not only leads to odour and taste problems but also poses a serious threat to fish and other aquatic organisms due to its highly toxic nature. Apart from this, some phenolic compounds also accelerate tumour formation leading to carcinogenicity. For these reasons, refinery waste-waters exceeding the limit of 0.05 mg/l of phenols are not permitted to be discharged into the main streams. In order to bring the concentrations of phenol within the permissible limit of the set standards, it becomes necessary to treat the refinery waste-water before it is discharged into the main stream.
For the removal of phenolic compounds from the industrial effluents, biological methods using single micro-organisms or a group of micro-organisms present in activated sludge have been used which utilize the organic compounds through a process called biodegradation (Rajakumar et al. 1991, Subhani et al. 1991). During biodegradation, the micro-organisms convert organic compounds into their inorganic forms which in turn serve as energy source or substrate for micro-organisms. The drawback of single bacteria is that it may not be able to fully biodegrade phenol and phenolic compounds due to non-availability of an array of enzymes responsible for complete biodegradation, wherein a microbial consortium proves to be better than a single bacterium for biodegradation because a number of enzymes present in different bacteria undergo co-metabolism. The micro-organisms present in the activated sludge have low efficiency because of suppression of specific bacteria present therein, thereby resulting in the inadequate and variable micro-flora and are not able to degrade the compounds of interest present in specific waste-waters. Another drawback of the conventional biological treatment method is that free microbial cells are unable to tolerate high concentrations of phenol present in petroleum refinery because of high toxicity of phenol on micro-organisms containing phenol degrading enzymes. Whereas, the advantages of the present invention over the above conventional methods envisage an effective and efficient removal of phenolic compounds from the waste-waters of petroleum refinery by selecting a blend of specialized micro-organisms from the source habitat which are capable of degrading them.
Though few micro-organisms have been immobilized on various supports to biodegrade phenol present in waste-waters, there are many drawbacks of the used supports as disclosed in the prior art. The supports are biodegradable, toxic, expensive, have low mechanical strength and have less surface area. Therefore, it is essential to develop microbial consortium, immobilized on a non-biodegradable, non-toxic, mechanically strong, having more surface area and economically cheaper support to enhance the biodegradability of phenol.
To overcome this problem, in the present invention, a defined microbial consortium is formulated as well as immobilized on non-biodegradable and economically cheaper support to achieve an effective as well as efficient biological treatment of phenolic waste-water of petroleum refinery. Immobilization of micro-organisms leads to a reduction in cell growth and protection of the microbial cells from the toxicant. Due to these reasons, immobilized cells offer a promising potential for an effective and efficient biological treatment of waste-waters. Immobilized bio-catalysts have also been used on an industrial scale for treating effluents containing phenolic compounds (Anselmo and Novail, 1992) as well as other toxicants (Wong et al, 1993 and Cijzen et al, 1988).
For solving the aforementioned problem, the applicants have realized that there exists a need to provide a process for an effective and efficient treatment of phenolic compounds present in petroleum refinery using microbial consortium immobilized on a non-biodegradable and economically cheaper support.
In the present invention, coconut fibre is used as a support for immobilization of microbial consortium having wider surface area of coconut fibres which enables adsorption of higher number of cells on the surface of the support which results in faster biodegradation of phenol. Coconut fibre as such is not easily biodegradable. Therefore, it has longer shelf life. This support is non-toxic to micro-organisms as well as is mechanically strong.
The main object of the present invention is to provide an immobilized microbial consortium and a process for the preparation of the said immobilized microbial consortium on a non-biodegradable and economically cheaper support for the treatment of phenolic waste-waters, which obviates the drawbacks as detailed above.
Another object of the present invention is to provide a process for the preparation of immobilized microbial consortium on non-toxic and mechanically strong support for the treatment of phenolic waste-waters.
Still another object of the present invention is to provide a process for the preparation of immobilized consortium on a support having wider surface area which enables the easy diffusion of oxygen as well as the nutrients which help in biodegradation of phenolic compounds.
The present invention relates to a process for the preparation of immobilized microbial consortium on coconut fibre for treatment of phenol present in petroleum refinery. Wider surface area of coconut fibres used for immobilization enables adsorption of higher number of cells on the surface of the support which results in faster biodegradation of phenol. Coconut fibre as such is not easily biodegradable. Therefore, it has a longer shelf life. The support is non toxic to micro-organisms as well as is mechanically strong.
The composition provided according to the present invention contains bacteria consisting of:
which facilitate effective and efficient treatment of phenol present in effluent of petroleum refinery. Above micro-organisms are deposited at Centre for Biochemical Technology Culture Collection (CBTCC) designated as stated above and will be made available to public on request as per normal official procedures.
The main characteristic features of all the bacterial cultures used for the invention which are similar to ATCC cultures are given below:
Characteristic Features of Aeromonas hydrophila (CBTCC/Micro/10)
Gram-negative
Motile by a single polar flagellum
Metabolism of glucose is both respiratory and fermentative
Oxidase positive
Catalase positive
Ferments salicin, sucrose and mannitol
Produces hydrogen sulphide from cysteine
Characteristic Features of Pseudomonas fluorescens (CBTCC/Micro/11)
Gram negative, aerobic rod shaped bacteria
Have polar flagella
Metabolism is respiratory, never fermentative
Catalase positive
Produces pyoverdin
Gelatin liquefaction positive
Characteristic Features of Pseudomonas aeruginosa (CBTCC/Micro/3)
Gram negative, aerobic rod shaped bacteria
Have polar flagella
Metabolism is respiratory, never fermentative
Catalase positive
Oxidase positive
Denitrification positive
Characteristic Features of Bacillus circulans (CBTCC/Micro/12)
Gram-positive, rod shaped
Forms endospores, resistant to many adverse conditions
Aerobic or facultative anaerobic
Motile
Utilizes acetate
Hydrolyzes starch
Characteristic Features of Yersinia enterocolitica (CBTCC/Micro/4)
Gram-negative
Facultative anaerobic, having both respiratory and fermentative type of metabolism
Oxidase negative
Motile
Produces acid from sucrose, cellobiose, sorbose and sorbitol
Characteristic Features of Enterobacter cloaca (CBTCC/Micro /1)
Gram-negative straight rods
Motile by peritrichous flagella
Facultative anaerobe
Ferments glucose with production of acid and gas
KCN and gelatinase positive
Nitrate reductase positive
Decarboxylates ornithine and arginine
Characteristic Features of Bacillus brevis (CBTCC/Micro/13)
Gram-positive, rod shaped
Forms endospores, resistant to many adverse conditions
Aerobic or facultative anaerobic
Motile
Grows at pH 6-8
Degrades tyrosine
The composition may contain the bacteria, in a preferred embodiment of the invention, in uniform amounts. The bacterial cultures of the above composition are isolated from the effluent of petroleum refinery. Effluent of petroleum refinery is collected from Mathura oil refinery. Effluent is homogenized and suspended in nutrient broth medium containing 0.1 gm/l of phenol concentration. Incubation is carried out for 16-24 hours, cultures are plated on Mac Conkey""s Agar. Colonies are mixed on a vortex mixer and all the cultures are isolated in pure cultures after several subcultures. The pure cultures are checked for the gram reactions. All the cultures are maintained as stock cultures.
The immobilization technique of formulated specialized microbial consortium of the present invention is carried out by inoculating the individual strains of the above mentioned bacteria separately in a nutrient broth. All the cultures are incubated preferably at 37xc2x0 C. For approximately 24 hours in an incubator shaker. For gentle shaking, the incubator shaker is maintained at an appropriate rpm, preferably at 75 rpm. After sufficient growth is obtained, the bacterial cells from these individual cultures are taken in the required quantity and mixed for preparing the microbial consortium. The resultant microbial consortium is centrifuged at appropriate rpm, preferably at 10,000 rpm for a period of approximately 30 minutes. The resultant pellet is washed by dissolving in minimum quantity of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at appropriate rpm, preferably at 10,000 rpm for a period of approximately 30 minutes. During centrifugation the temperature is maintained preferably at 4xc2x0 C. The resultant pellet was suspended in 2% of 25% (v/v) solution of glutaraldehyde for 2 hours at 100 rpm preferably at 35xc2x0 C. to 37xc2x0 C. The suspension of bacterial culture was centrifuged at appropriate rpm, preferably 10,000 rpm for a period of approximately 30 minutes. The resultant pellet was washed by dissolving in phosphate buffer, 0.05 M, pH 6.8 and centrifuging at approximate rpm, preferably at 10,000 rpm for a period of 30 minutes. The resultant pellet was suspended in 50 ml phosphate buffer, 0.05 M, pH 6.8. The support for immobilization was prepared by cutting the coconut fibre in small pieces having 1.5 inch length, 1.5 inch breadth and 1.0 inch diameter. The pre-treatment of pieces of coconut fibre was carried out by dipping in 1.0% v/v solution of HgCl2 for a period of 5-10 minutes. The treated coconut fibre was removed and washed thoroughly with running tap water followed by triple distilled water. The obtained treated pieces of coconut fibres were dried in oven at around 40xc2x0 C.-50xc2x0 C. and sterilized by autoclaving at 15 lbs for 15 minutes. The resultant sterilized pieces of coconut fibre were dipped into the already prepared bacterial suspension in phosphate buffer, 0.05 M, pH 6.8 and incubated at appropriate temperature, preferably at 37xc2x0 C. After proper incubation, the dipped pieces of coconut fibre were removed from the bacterial suspension. The obtained immobilized coconut fibres were air dried for 4-6 hours and stored at appropriate temperature preferably at 4xc2x0 C. The immobilized pieces of coconut fibre so obtained are used for the efficient and effective treatment of synthetic phenol as well as phenol present in the industrial effluent of petroleum refinery.
Accordingly, the present invention provides a process for the preparation of microbial consortium immobilized on a non-biodegradable and economically cheaper support for the treatment of phenolic waste-waters which comprises of:
The invention further provides for the preparation of immobilized microbial consortium which comprises:
(a) isolating a range of bacterial strains from effluent collected from petroleum refinery industry.
(b) culturing the said bacterial strain on nutrient media to get pure cultures.
(c) inoculating the said bacterial strains in nutrient broth, individually.
(d) incubating the said bacterial strains and growing the said incubated strains, individually;
(e) testing the phenol tolerance ability of the said grown bacterial cultures;
(f) selecting the said bacterial strains showing phenol tolerance limit for 0.2 to 0.4 gm/l;
(g) acclimatizing the said selected bacterial strains;
(h) mixing the said, acclimatized bacterial strains in equal proportions on the basis of optical density values to formulate a microbial consortium;
(i) centrifuging the said mixed bacterial strains to obtain pellet, washing the collected pellet with phosphate buffer, re-centrifuging, obtaining pellet;
(j) suspending the said pellet in 2% of 25% (v/v) solution of glutaraldehyde, a non-toxic cross-linking agent, for 2 hours at 75-100 rpm at a temperature ranging from 30-37+ C.;
(k) centrifuging the suspended bacterial cultures to obtain pellet, washing the obtained pellet with phosphate buffer 0.05-0.2 M, pH 6.8-7.2, re-centrifuging, obtained pellet;
(l) preparing the support by cutting the coconut fibre in small pieces;
(m) dipping the coconut fibre pieces in 0.05%-0.2% v/v solution of HgCl2 for a period ranging from 5-10 minutes, washing thoroughly with triple distilled water;
(n) drying the coconut fibre pieces in oven and sterilizing;
(o) suspending the obtained pellet as in step (k) in phosphate buffer 0.05 M, pH 6.8, dipping the treated coconut fibre pieces from step (n);
(p) incubating the said bacterial suspension containing coconut fibres at 30-37xc2x0 C. overnight;
(q) discarding the suspension, collecting the coconut fibre pieces on which micro-organisms are immobilized;
(r) air drying the coconut fibre pieces;
(s) storing the air dried coconut fibre pieces preferably at 1-4xc2x0 C.;
(t) testing the air dried coconut fibre pieces for biodegradation of synthetic phenol;
In an embodiment of the present invention, the formulated microbial consortium is obtained by inoculating a suspension of the bacteria selected from a group consisting of Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus circulans, Yersinia enterocolitica, Enterobacter cloaca, and Bacillus brevis. 
In another embodiment of the present invention, the individual strains of above mentioned bacteria are inoculated separately in nutrient broth.
In yet another embodiment of the present invention, incubation is carried out by gentle agitation at approximately 75-100 rpm.
In one of the embodiment of the present invention, the growth of the incubated bacterial strains is carried out at temperature ranging between 30xc2x0 C. to 37xc2x0 C. For a period of 16-24 hours.
In another embodiment of the present invention, the grown, pure bacterial cultures were tested for phenol tolerance ability, by growing in phenol concentrations ranging from 0.2 to 1.0 g/l.
In an embodiment of the present invention, the said pure bacterial cultures tolerating phenol concentration up to 0.4 g/l were selected.
In a further embodiment of the present invention, the selected said bacterial strains were acclimatized to a higher concentration of phenol ranging from 0.5-2.0 g/l.
In still another embodiment of the present invention, the said individual, acclimatized bacterial strains are mixed in equal proportions.
In a further embodiment of the present invention, the resultant microbial consortium is centrifuged at appropriate rpm preferably at 8000-12000 rpm for a period of approximately 20-30 min.
In another embodiment of the present invention, the resultant pellet of microbial consortium is washed by dissolving in a appropriate quantity of phosphate buffer 0.05 M-0.1M, and pH 6.8-7.2 and re-centrifuging at an appropriate rpm in the range of 8000-12000 for a period of approximately 20-30 min., at a temperature ranging from 1 to 4xc2x0 C.
In an embodiment of the present invention, the obtained pellet of microbial consortium suspended in 2% of 25% (v/v) solution of gluteraldehyde for 2 hours at 100 rpm preferably at 30xc2x0 C. to 37xc2x0 C.
In a further embodiment of the present invention, centrifuging the suspended microbial consortium to obtain the pellet, washing the obtained pellet with phosphate buffer 0.05 M-0.2M, and pH 6.8-7.2, recentrifuging, obtaining pellet.
In still another embodiment of the present invention, the support is prepared by cutting the coconut fibre in small pieces, dipping in 0.05%-0.2% v/v of HgCl2 for 5-10 minutes, washing thoroughly in running tap water, again washing with triple distilled water, drying in oven and sterilizing the coconut fibre pieces at 15 lbs for 15-30 minutes. In another embodiment of the present invention, suspending the obtained pellet of microbial consortium in phosphate buffer, 0.05 M-0.2 M, pH 6.8-7.2.
In still another embodiment of the present invention, the treated coconut fibre pieces are dipped into the suspension of the microbial consortium.
In a further embodiment of the present invention, the inoculated coconut fibre pieces are incubated at a temperature ranging between 30xc2x0 C. to 37xc2x0 C. For a period of 16-24 hours.
In another embodiment of the present invention, the incubated coconut fibre pieces are separated from the suspension of microbial consortium.
In still another embodiment of the present invention, the separated coconut fibre pieces are air-dried for 4-6 hours and stored at temperature ranging between 1xc2x0 C. to 4xc2x0 C.
In a further embodiment of the present invention, the formulated microbial consortium, immobilized on coconut fibre pieces is used for the biodegradation of synthetic phenol ranging between 0.6-2.0 g/l.
The examples provided below are given by way of illustration of the invention and therefore, should not be construed to limit the scope of invention.